PEDS Advance Access published online on September 7, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm045
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Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure
1Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan
3 To whom correspondence should be addressed. E-mail: nagamune{at}bio.t.u-tokyo.ac.jp
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Abstract |
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Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron–sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min–1) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.
Keywords: branched structure/cytochrome P450/CYP101/site-specific cross-linking/transglutaminase
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Introduction |
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TopAbstractIntroductionMethodsResults and discussionConclusionReferences |
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Cytochrome P450s (P450s) are heme-containing monooxygenases involved in various physiological processes from archaea to mammals (Ortiz de Montellano, 1995
). They catalyze diverse types of reactions (Sono et al., 1996) containing hydroxylations, epoxidations, dealkylations and heteroatom oxidations, which make P450s attractive industrial catalysts (Martinez and Stewart, 2000). P450s require electrons from NAD(P)H to convert molecular oxygen into reactive species and are classified by electron transfer manner. Class II P450s, which locate on the microsome membrane, receive electrons from membrane-binding flavoproteins (P450 reductases), whereas class I P450s, which are bacterial and mitochondrial P450, receive electrons from a flavoprotein via an iron–sulfur protein. Bacterial P450s are soluble proteins and more appropriate for industrial catalysts than other P450s and a lot of studies toward practical applications of bacterial P450s have been reported (Reipa et al., 1997; Wong et al., 1997; Urlacher and Schmid, 2002; Cryle et al., 2003). However, excess amounts of these proteins are necessary to keep enough concentration of reduced electron transfer proteins for sufficient reaction rates and coupling efficiencies (Kadkhodayan et al., 1995) and these properties are bottlenecks for practical applications of P450s. The P450 from Pseudomonas putida (P450cam) that catalyzes stereoselective hydroxylation of d-camphor is a well-studied cytochrome P450 as a model P450 for reaction mechanisms (Brewer and Peterson, 1988; Schlichting et al., 2000; Makris et al., 2002; Purdy et al., 2004) and structural studies (Poulos et al., 1987; Hays et al., 2004; Nagano and Poulos, 2005). The catalytic turnover of P450cam requires two electron transfer-related proteins, the iron–sulfur protein putidaredoxin (Pdx) and the flavoprotien Pdx reductase (Pdr). Pdr accepts two electrons from NADH, and Pdx transports one electron at a time from Pdr to P450cam. Although one P450cam only requires one molecule each of Pdx and Pdr stoichiometrically, excess amounts, i.e. non-catalytic amounts of Pdx and Pdr are required to attain the maximum activity (Kadkhodayan et al., 1995). A fusion protein composed of these three component proteins could achieve an extremely high local concentration, and also bring a high catalytic activity owing to the increase in the effective concentration. Furthermore, this could bring a high coupling efficiency because catalytic amounts of Pdx and Pdr minimize the reoxidation of these proteins by molecular oxygen as well. In general, fusion proteins can be prepared by (i) a genetic method, in which the component proteins are genetically linked and expressed as a single polypeptide chain, and (ii) a chemical method, in which the component proteins are cross-linked by chemical reagents after individual expression of each protein. To date, the former method has not yet provided sufficiently active fusion proteins due to the structural constraints (Black et al., 1994; Sibbesen et al., 1996), whereas the latter method is hard to control the cross-linking sites in general.
To construct a new type of P450 fusion protein, we utilized an enzymatic method involving a transglutaminase (TGase). TGases catalyze the formation of an 
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-glutamyl)lysine bond between the side chains of a glutamine residue and a lysine residue. The TGase from Streptomyces mobaraensis, which is widely used in food industry (Yokoyama et al., 2004), has been reported to recognize the sequence around the F-helix of horse heart myoglobin (HEAELKPLAQSHATKHKIPIK, reactive residues shown in bold) (Takazawa et al., 2004) and catalyzed site-specific cross-linking of tagged proteins with the recognition sequence under protein-friendly conditions (Kamiya et al., 2003; Takazawa et al., 2004; Tanaka et al., 2004, 2005). Such TGase-mediated site-specific protein cross-linking enables the formation of a branched fusion protein with spatially equal geometry of the three component proteins, which should result in fewer structural constraints than a tandem linear fusion protein, as well as an intramolecular electron transfer (Fig. 1). Development of genome analysis has revealed many bacterial P450 gene sequences, although every electron transfer proteins for each P450 have not been discovered and few electron transfer proteins have been characterized. Newly discovered P450s have been characterized using Pdx and Pdr (Koo et al., 2000; Grogan et al., 2002); however, some P450s cannot show the catalytic activity (Ke et al., 2005) probably due to the low efficiency of electron transfer. Applying foreign P450s to a branched fusion protein that includes Pdr and Pdx would be useful to characterize various P450s and be able to use themselves as self-sufficient oxygenases for industry. Here, we report creation of a novel artificial P450 fusion protein with a site-specific branched structure toward efficient intramolecular electron transfer via this fusion protein.
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Methods |
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Expression and purification of proteins
Construction of vectors is described in supplementary methods available at PEDS online. A fusion protein between Pdr and P450cam, which is cross-linked with a peptide including a reactive glutamine residue for TGase, Pdr–Qlinker–P450cam (Fig. 2) was expressed from pEQCen in Escherichia coli BL21(DE3) by innoculating a single colony of cells in 1 l of Terrific Broth (TB) containing 100 µg ml–1 ampicillin and 1 mM 5-aminolevulinic acid hydrochloride (Cosmo Bio) and cultivating cells at 27°C overnight. The cells were harvested by centrifugation and disrupted by sonication in 50 ml of buffer A (50 mM potassium phosphate buffer, pH 7.4, containing 500 mM KCl, 40 mM imidazole, 5 mM d-camphor and 10% glycerol) containing 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF: Sigma) and 33 units ml–1 Benzonase (Sigma). The lysate was centrifuged (22 000g, 30 min) at 4°C and then purified with a HisTrap HP column (1.6 x 2.5 cm: Amersham Biosciences). After buffer exchange for buffer B (5 mM potassium phosphate buffer, pH 7.4, containing 5 mM d-camphor) with a HiTrap Desalting column (1.6 x 2.5 cm: Amersham Biosciences), the isolated protein was treated with enterokinase (Invitrogen) at 20°C for overnight. The cleaved protein was applied to a HisTap HP column and eluted with buffer A. After the buffer exchange for buffer C (50 mM potassium phosphate buffer, pH 7.4, containing 5 mM d-camphor) with a HiTrap Desalting column, anion exchange chromatography was carried out on a HiTrap DEAE FF column (1.6 x 2.5 cm: Amersham Biosciences) with a 0–400 mM KCl gradient. After concentration by ultrafiltration using an Amicon Ultra-15 PLQK (Millipore), the protein was subjected to gel-filtration chromatography on a Superdex 200 HR 10/30 column (1 x 30 cm: Amersham Biosciences) with buffer D (50 mM potassium phosphate buffer, pH 7.4, 150 mM KCl, 5 mM d-camphor). The purified protein was stored at –80°C until use. Pdr–Alinker–P450cam and Qtag–P450cam (Fig. 2) were expressed and purified as described earlier. His6-P450cam WT and His6-P450cam mutant were expressed and purified as described earloer except for the cleavage by entreokinase and the second affinity purification step.
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Pdr–Qtag (Fig. 2) was expressed in E.coli BL21(DE3) pLysS transformed with pERQen. After growing cells from a single colony in 5 ml of LB containing 50 µg ml–1 ampicillin and 34 µg ml–1 chloramphenicol at 37°C overnight, the cells were added to 1 l of TB containing 100 µg ml–1 ampicillin and 34 µg ml–1 chloramphenicol and cultivated at 37°C. When the OD600 reached a value of 0.6, 100 mg of ampicillin was added and the temperature was lowered to 27°C. After overnight culture, the cells were harvested by centrifugation. The purification was conducted as described for Pdr–Qlinker–P450cam, except that buffers did not contain d-camphor and a Superdex 75 HR 10/30 column (1 x 30 cm: Amersham Biosciences) was used instead of a Superdex 200 HR 10/30 column. Wild-type Pdr was expressed as described for Pdr–Qtag and purified as described previously (Sevrioukova et al., 2001
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The Cys73Ser/Cys85Ser mutant of Pdx (abbreviated as C73S/C85S Pdx) was expressed and purified as previously described (Sevrioukova et al., 2003). Pdx–CKtag (Fig. 2) was expressed in E.coli BL21(DE3) pLysS transformed with pEXK. After growing cells in 5 ml of LB containing 50 µg ml–1 ampicillin and 34 µg ml–1 chloramphenicol at 37°C overnight, the cells were added to 1 l of TB containing 100 µg ml–1 ampicillin and 34 µg ml–1 chloramphenicol, and cultivated at 37°C. When the OD600 reached a value of 0.6, 100 mg of ampicillin and 1 mmol of IPTG were added and the temperature was lowered to 27°C. After growing for overnight, the cells were harvested by centrifugation and disrupted by sonication in 50 ml of buffer E (50 mM potassium phosphate buffer, pH 7.4, 500 mM KCl, 40 mM imidazole, 10% glycerol) containing 0.1 mM AEBSF and 33 units ml–1 Benzonase. The lysate was centrifuged (22 000g, 30 min) at 4°C and then purified using a HisTap HP column. After buffer exchange for buffer F (50 mM potassium phosphate buffer, pH 7.4) with a HiTrap Desalting column, anion exchange chromatography was carried out on a HiTrap DEAE FF column with a 0–400 mM KCl gradient. After concentration by ultrafiltration using an Amicon Ultra-15 PLQK, the protein was subjected to gel-filtration chromatography on a Superdex 75 HR 10/30 column with buffer G (50 mM potassium phosphate buffer, pH 7.4, containing 150 mM KCl). The purified protein was stored at –80°C until use. Pdr–CAtag (Fig. 2) was expressed and purified in a similar manner.
TGase from S.mobaraensis (Ajinomoto) was purified as follows. The enzyme was dissolved in buffer G and subjected to gel-filtration chromatography on a Superdex 75 HR 10/30 column with buffer G. The isolated enzyme was further purified using a HisTrap HP column. After buffer exchange for buffer G with a HiTrap Desalting column, the enzyme was stored at –80°C until use.
Preparation of site-specific cross-linked proteins
A mixture of 48 µM Pdr–Qlinker–P450cam and 49 µM Pdx–CKtag were incubated with 1 µM TGase in 4 ml of buffer D at 2°C overnight. The reaction solution was subjected to affinity chromatography (HisTrap HP column) and gel-filtration chromatography (Superdex 200 HR 10/30 column) to obtain purified fusion protein (designated bRXC for branched Pdr–Pdx–P450cam triple fusion protein). Pdr–Qtag–Pdx and Pdx–Qtag–P450cam were prepared with Pdr–Qtag and Qtag–P450cam, respectively, as a substitution of Pdr–Qlinker–P450cam as described earlier, except that a Superdex 75 HR 10/30 column was used instead of a Superdex 200 HR 10/30 column. The concentrations of heme-containing proteins were determined by the pyridine hemochromogen method (Omura and Sato, 1964). The concentrations of the other proteins were determined by BCA method.
Molecular mass was measured by means of a matrix-assisted laser desorption ionization (MALDI-TOF) system, Voyager System 4338 from Applied Biosystems. Sinapinic acid was used as a matrix, and bovine serum albumin was used as a standard.
Dissociation constant determination
Dissociation constant (Kd) for d-camphor and bRXC complex was determined at 25°C by titration of substrate-free bRXC (1.0 µM) with d-camphor in the range of 0.1 to 20 µM in buffer G. Binding was followed by monitoring the decrease in absorbance at 418 nm.
Catalytic activities were determined from the difference between the rates of NADH oxidation by measuring absorbance of NADH at 340 nm (340 = 6.22 mM–1 cm–1) at 25°C with and without d-camphor. Typical reaction mixtures contained 0.05 µM proteins, 250 µM d-camphor and 90 µM NADH in 2 ml of buffer G. Reactions were initiated by adding proteins which contain a P450cam moiety. Ferricyanide reduction activity was measured spectroscopically at 420 nm (420 = 1.02 mM–1 cm–1) in buffer G containing 40 µM potassium ferricyanide and 50 µM NADH. Cytochrome c reduction activity was measured spectroscopically at 550 nm in buffer G containing 20 µM cytochrome c and 50 µM NADH. The extinction difference (cytochrome cred – cytochrome cox at 550 nm) used to calculate the activity was 21 mM–1 cm–1.
Coupling efficiencies were determined from the ratio of d-camphor consumption to NADH consumption. The reaction mixtures contained 0.05 µM bRXC, 2.0 mM d-camphor and 1.0 mM NADH in 800 µl of buffer G. Reactions were conducted at 25°C for 180 min. 3-Endo-bromocamphor was added as an internal standard and d-camphor was immediately extracted 5 times with 200 µl of C2H4Cl2. After removal of water with anhydrous magnesium sulfate, the extract was concentrated under a N2 flow and analyzed by gas chromatography. Gas chromatography analysis was performed using a Hewlett-Packard 6850 equipped with a CHRALDEX-GTA capillary column (25 m x 0.25 mm I.D.; Advanced Separation Technology) programmed to run at 160°C for 5 min. NADH in the reaction mixture was recovered from the water layer after extraction of the d-camphor and the concentration was estimated from the absorption at 340 nm.
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Results and discussion |
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Our preliminary experiments revealed that wild-type P450cam contains reactive glutamine and lysine residues for TGase, whereas Pdr and C73S/C85S Pdx do not have reactive residues (data not shown). Every potentially exposed glutamine and lysine residues (Gln7, Gln211, Gln214, Lys215, Gln273, Gln312, Lys314, Lys315, Gln344, Lys345, Gln389, Gln391 and Lys413) were substituted with asparagine and arginine residues, respectively, because clear consensus amino acid sequence in substrates for TGase has not been identified. On the basis of the crystal structure of P450cam, all the residues mentioned above are not expected to be located near the proximal residues which can be involved in the electron transfer and interaction between Pdx and P450cam. Pdr–Qlinker–P450cam was constructed by genetically fusing the C-terminus of Pdr and the N-terminus of P450cam with a specific peptide linker for TGase containing a reactive glutamine residue (Q-linker), whereas Pdx–CKtag was constructed by genetically fusing a specific peptide linker for TGase containing a reactive lysine residue (CK-tag) to the C-terminus of a Cys73Ser/Cys85Ser mutant of Pdx (Fig. 2). We evaded linking the N-terminus of Pdr and the C-terminus of P450cam because an additional peptide linker at the N-terminus inactivates Pdr and that at the C-terminus dose not inactivate (Sevrioukova and Poulos, 2002
) and (His)6 tag at N-terminus dose not interfere with the reactivity of P450cam toward Pdx or O2 (Purdy et al., 2004). A crystal structure of Pdx shows that the C-terminus of Pdx is parallel to the exposed direction of a loop including Cys39 and Cys 45 which are binding to the [2Fe–2S] cluster and the N-terminus of Pdx locates at the opposite side of [2Fe–2S] cluster (Sevrioukova et al., 2003). We chose the C-terminus as a linking position based on our expectation that the [2Fe–2S] cluster could exclusively face the center of the fusion protein to prevent intermolecular electron transfer, whereas conjugation of Pdx to Pdr–Qlinker–P450cam at N-terminus would be expected to face the cluster outwards and subsequently decrease probability of interactions between Pdx and the other domains intramoleculary. These proteins were expressed and purified separately. The TGase-catalyzed reaction of Pdr–Qlinker–P450cam and Pdx–CKtag yielded a site-specific cross-linked fusion protein, bRXC, from equal molar amounts of Pdr–Qlinker–P450cam and Pdx–CKtag (Fig. 3). The reaction yield was 80% on SDS–PAGE analysis (data not shown). MALDI-TOF/MS analysis revealed that bRXC (110 768 Da) was composed of one molecule of Pdr–Qlinker–P450cam (96 124 Da) and one molecule of Pdx–CKtag (14 777 Da).
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The extinction coefficients of Pdr–Qlinker–P450cam, His6-P450mutant and Qtag–P450cam at 392 nm in the presence of d-camphor were 90.9, 91.0 and 99.2 mM–1 cm–1, respectively. On the basis of the concentrations determined by BCA assay, those of Pdx–CKtag at 412 nm and Qtag–Pdr at 455 nm were calculated to be 7.0 and 14.5 mM–1 cm–1, respectively. bRXC showed the typical UV–Vis spectrum of a high spin state of P450cam moiety in the presence of a substrate (
392 = 115.8 mM–1 cm–1), and the ferrous CO-complexed state did not show a peak at 420 nm (Fig. 4a) which is the characteristic absorption peak of the inactive form of P450s (Martinis et al., 1996). The Kd value for d-camphor–bRXC complex was estimated to be 0.80 ± 0.05 µM with spectral titration, and that for d-camphor–wild-type P450cam complex is reported to be 0.79 ± 0.02 µM (French et al., 2001). These results indicate that the heme domain of bRXC was properly folded and was active. bRXC also showed an absorption peak at 455 nm derived from Pdr (Fig. 4b). A linear combination of the component protein spectra (Pdr–Qtag + Qtag–P450cam + Pdx–CKtag) was similar to the spectrum of bRXC (Fig. 4c). This result suggests that Pdr–Qlinker–P450cam contains both cofactors, heme and FAD, and the TGase-catalyzed cross-linking between Pdr–Qlinker–P450cam and Pdx–CKtag dose not lose the cofactors. Therefore, it was likely that the enzymatic cross-linking does not damage native function and/or structure of each protein component in bRXC.
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bRXC showed d-camphor-dependent catalytic activity without addition of Pdr, Pdx or P450cam. A Lineweaver-Burk plot (Fig. 5) indicated that the reaction followed in a ping-pong mechanism and that the Km values for NADH and d-camphor were 2.5 ± 0.1 and 1.3 ± 0.1 µM, respectively, under saturation conditions of the alternate substrate. This Km value for d-camphor is similar to that of wild-type P450cam (1.6 µM) (French et al., 2001
). The activity of bRXC was higher than that of the P450cam system reconstituted with equimolar concentrations of the three component proteins (Fig. 6). The initial rate increased by a factor of 31 when the protein concentration was 0.2 µM. The activity of bRXC increased in a first order manner depending on the concentration, whereas that of the reconstituted P450cam increased in a higher order manner. Furthermore, any breakages of the cross-links in bRXC caused critical loss of its activity (Fig. 7). These facts indicate that electrons are transferred within the molecule and that this intramolecular electron transfer process dominates the activity enhancement. The catalytic activity of bRXC was calculated to be 306 ± 3 min–1 from the linear relationship between NADH oxidation rate and bRXC concentration (Fig. 6), which is 10-fold larger than that of the previously reported tandem linear fusion P450cam (30 min–1) (Sibbesen et al., 1996). This difference should be attributed to the novel branched structure, in which Pdx is linked at one point in bRXC compared with two points in the linear fusion protein.
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The activity of bRXC did not reach at the maximum activity of a reconstituted P450cam system, which was reported to be 2.41 x 103 min–1 under a saturation condition ([Pdr] = 4 µM, [Pdx] = 10 µM) (Kadkhodayan et al., 1995
). This indicates a possibility of further improvements in the catalytic activity of bRXC. The rate constant of a reconstituted P450cam system with the C73S/C85S Pdx under a saturation condition ([Pdr–Qtag] = 4 µM, [C73S/C85S Pdx] = 80 µM) was 1.91 x 103 min–1 (Fig. 8). Compared with a previous report (Kadkhodayan et al., 1995), the saturation concentration of Pdx was increased 8-fold and the activity of P450cam under that condition was decreased to 79%. This result is consistent with the report that the mutation C73S/C85S in Pdx partly decreased the electron transfer activity, although it markedly improved the stability of Pdx (Sevrioukova et al., 2003).
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It is reported that Trp106 at the C-terminus of Pdx is not important for the electron transfer from Pdr to Pdx and Trp106 prevents tight binding of Pdx to Pdr (Kuznetsov et al., 2005
), whereas Trp106 is reported to be important for the electron transfer to P450cam (Stayton and Sligar, 1991; Kuznetsov et al., 2006). In fact, Pdx–CKtag showed a much lower affinity for P450cam than C73S/C85S Pdx (Fig. 8). The Km for Pdx–CKtag could not be determined due to its low affinity, whereas the Km for C73S/C85S Pdx was 13 ± 1 µM. This result indicates that an additional tag at C-terminus of Pdx might have negative effects on the electron transfer between P450cam and Pdx. Reduction assay of artificial electron transfer acceptors is useful to monitor electron flow through the individual parts of bRXC. Ferricyanide can be reduced by both Pdr and Pdx, although the reduction rate of ferricyanide by Pdr is much faster than that of Pdx by Pdr (Sevrioukova and Poulos, 2002). If it is assumed that electron transfer to ferricyanide via Pdx moiety from Pdr moiety in bRXC is negligible and ferricuanide is directly reduced by Pdr moiety, the results summarized in Table I suggest that bRXC mostly retains its electron transfer ability from NADH. Both Pdr and Pdx can also transfer electrons to cytochrome c; however, direct cytochrome c reduction by Pdr moiety in bRXC is negligible because the rate of cytochrome c reduction by Pdr is much slower than that by reduced Pdx. It is possible to evaluate electron flow from Pdr moiety to Pdx moiety in bRXC by comparing cytochrome c reduction activity of bRXC to that of Pdx in the presence of an excess Pdr concentration (Fig. 9). Cytochrome c reduction activity of bRXC (459 mol/min/mol of bRXC) is 71% of that of C73S/C85S Pdx in the presence of 4 µM Pdr (649 mol/min/mol of Pdx) (Table I), whereas cytochrome c reduction rate by wild-type Pdx in the presence of 4 µM Pdr is reported to be 365 mol/min/mol of Pdx (Sibbesen et al., 1996). This suggests that electron transfer from Pdr moiety to Pdx moiety in bRXC is not a dominant step to decrease the activity of bRXC to 16% of the reconstituted P450cam system under a saturated condition. Therefore, it is likely that the presence of an additional C-terminal sequence of Pdx–CKtag disturbs the intramolecular interaction between Pdx and P450cam moieties of bRXC. Nevertheless, this disturbance by an additional C-terminal sequence was overcome by the proximity effect based on the bRXC structure, although optimization of the peptide linkers and protein arrangement may further increase the activity.
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The broad substrate specificity and a wide variety of reactions of P450s attract the attention for their application as industrial biocatalysts for organic synthesis. However, the requirement for excess amounts of redox partners is a bottleneck for practical applications. Recently, new classes of P450, which contains a heme domain and electron transfer domains within a molecule, have been reported (De Mot and Parret, 2002
). Class III P450 is naturally occurred fusion proteins of class II P450 and P450 reductase (Nakayama et al., 1996; Ost et al., 2001; Munro et al., 2002; Gustafsson et al., 2004), and class IV P450 is composed of a heme domain, FMN binding domain, NADH binding domain and iron–sulfur cluster (Roberts et al., 2002, 2003). P450 from Bacillus megaterium (P450 BM3), which is classified into class III P450, shows highest catalytic activity due to efficient electron transfer (Munro et al., 2002). From this result, a fusion protein of bacterial class I P450 and its electron transfer proteins are expected to have high catalytic activity. In fact, bRXC, which can work without excess amounts of Pdx and Pdr, showed substantial catalytic activity as we anticipated.
With respect to the coupling efficiency (consumed d-camphor/consumed NADH), bRXC showed comparable efficiency (99%, Table II) with the reconstituted P450cam system under the saturation conditions (96%) (Kadkhodayan et al., 1995). This indicates that efficient electron transfer occurred in bRXC even in the absence of excess amounts of Pdx and Pdr. P450 BM3, which contains equimolar amounts of the heme domain and electron transfer domains within a single polypeptide chain, similar to bRXC, is the most catalytically active P450 and its coupling efficiency was reported to be 91% (Ost et al., 2001). Therefore, bRXC, an artificial P450 consisting of a branched single polypeptide chain, has a potency comparable with the naturally occurring single polypeptide P450 BM3.
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Conclusion |
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TopAbstractIntroductionMethodsResults and discussionConclusionReferences |
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A novel artificial P450 fusion protein, which shows high catalytic activity and coupling efficiency via efficient intramolecular electron transfer, was successfully created by TGase-catalyzed site-specific protein cross-linking. The branched structure simply increases the concentrations of the redox partners for P450 without causing a large loss of freedom of the proteins, and this method is therefore applicable for other soluble P450s including P450cam mutants. P450cam in bRXC can also be substituted to other P450s because the electron transfer system consisting of Pdr and Pdx can transfer electrons to other soluble P450s (Shimoji et al., 1998
; Koo et al., 2000; Grogan et al., 2002; Xu et al., 2005). Therefore, this branched P450 system will enable the specific oxidations of a wide range of substrates and promote studies on novel P450s, whose redox partners have not yet been identified or are unstable. TGase-catalyzed protein cross-linking is limited to proteins of which surface lysines and glutamines, which could be important for activity and/or stability, are not recognized. To apply this method to other P450s, it is necessary to develop a new preparation of TGase-mediated site-specific branched fusion P450 independent of reactive lysines and glutamines on surface of P450s because bacterial TGase has rather broad substrate specificity (Bechtold et al., 2000; Sato et al., 2001). This branched fusion method is also applicable to other multicomponent enzymes such as Rieske non-heme iron dioxygenases (Wackett, 2002). The TGase-mediated branching of chimeric proteins can offer a breakthrough for kinetic studies and practical uses of multicomponent enzymes as well as P450s.
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Footnotes |
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Abbreviations: His6-P450cam WT, His6-tagged P450cam at the N-terminus of the wild-type P450cam; His6-P450cam mutant, His6-tagged P450cam at the N-terminus of a mutant P450cam; C73S/C85S Pdx, the Cys73Ser/Cys85Ser mutant of Pdx; Qlinker, a peptide sequence including a reactive glutamine residue for TGase; CKtag, a peptide sequence including a reactive lysine residue for TGase, Pdr–Qlinker–P450cam, a fusion protein between Pdr and P450cam with Qlinker; Pdx–CKtag, Pdx fused with CKtag at the C-terminus of C73S/C85S Pdx; bRXC, branched Pdr–Pdx–P450cam triple fusion protein.
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Acknowledgment |
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We thank Prof Goto and Dr Ichinose of Kyushu University for plasmids encoding the genes for putidaredoxin and putidaredoxin reductase and Prof Sligar of University of Illinois for a plasmid encoding the gene for P450cam. We are also grateful to Ajinomoto Co., Inc., for providing the TGase sample. This work was partly supported by the 21st century COE program ‘Human-Friendly Material Based on Chemistry’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Received April 9, 2007; revised July 17, 2007; accepted July 17, 2007.
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